Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these references is incorporated herein as though set forth in full.
Mutagenic lesions in DNA frequently result from structural modifications of the heterocyclic bases (exocyclic adducts, free radical-induced modifications) and/or from complete removal of the base (abasic sites). Further, cellular processing of lesion containing DNA can lead to mismatches, additions, deletions or base pair substitutions. Thus, by lesion, it is meant to include mismatches, additions and deletions. A wide range of lesion-induced thermodynamic effects have been observed. Typically, the free energy stabilizing the duplex is reduced significantly by inclusion of the lesion. Methods of determining lesion effects on duplex free energy are limited. Typically, the effect of a DNA modification on the energetics of duplex formation is measured by comparison of independently measured association constants for the modified and unmodified duplex or by comparing Tm values, which are commonly but erroneously believed to represent thermodynamic stability. Therefore, there is a need for a simple, reproducible and sensitive method for rapidly screening for duplex stability.
Typically, the effect of a DNA modification on the energies of duplex formation have been measured by comparison of independently measured association constants for the modified DNA and the unmodified duplex requiring two separate experiments.
Furthermore, there is a large technical barrier for direct measurement of single duplex association events. In conventional titration experiments, a solution of one strand is added to a solution of its complement with formation of a duplex monitored by any of a variety of methods, including spectroscopic and calorimetric methods. To extract useful information from a conventional titration, the experiment must be devised such that a significant fraction of free titrant will be present throughout the titration. Satisfaction of this condition leads to the familiar sinusoidal shape of the titration curve. To satisfy this condition, typically the product of the initial titrate concentration, c, and the association constant, K, is in the range 10<cK<1000. Due to the high association constant for nucleic acid duplexes, the component concentration must be below the association constant, accordingly, the components are likely to be too dilute to be detected by standard spectroscopic means.
A common spectroscopic method for monitoring duplex formation relies on the hyperchromicity of duplex formation. However, the extinction coefficients of duplexes of similar length is not a very sensitive reporter of the small differences in DNA content that are of most interest, as in the case of oligonucleotide duplexes with damage to only a single base. Even a technique such as circular dichroism is not sufficiently sensitive and also suffers from difficulties in interpretation of spectral variation which can be due to factors other than duplex formation.